Hepatitis C virus (hereinafter referred to as `HCV`) is a major etiologic agent for the post transfusional and sporadic non-A, non-B hepatitis which develops into hepatocellular carcinoma through chronic hepatitis and liver cirrhosis in more than 50% of the cases (see: Alter, H. J., Transfusion-associated non-A, non-B hepatitis: the first decade, In A-J Zuckerman Ed. (1988); Choo Q. L. et al., Science, 244:359-362 (1989); Choo Q. L. et al., British Medical Bulletin, 46 (2):423-441 (1990)). Considering its seriousness due to high mortality and morbidity, much interests have been centered on the development of a diagnostic reagent and a vaccine for HCV infection. Although much progress has been made at the molecular level since a whole HCV genome was cloned and nucleotide sequences were analysed (see: Choo Q. L. et al., Proc. Natl. Acad. Sci., USA, 88:2451-2455 (1991); Kato, N. et al., Proc. Natl. Acad. Sci., USA, 87:9524-9528 (1990)), the difficulty of obtaining enough free viruses either from in vitro propagation or blood of HCV infected patient has been a barrier in development of conventional vaccine including whole killed or attenuated viral vaccine. Under the circumstances, heterologous expression system has been considered to be the best choice to obtain useful HCV antigens as components of HCV subunit vaccine.
On the other hand, HCV has a 9.4 kb single-stranded positive sense RNA genome which encodes a single polyprotein of about 3,010 amino acids (see: Choo Q. L. et al., Proc. Natl. Acad. Sci., USA, 88:2451-2455 (1991); Takamizawa, A. et al., J. Virol., 65:1105-1113 (1991); Kato, N. et al., Proc. Natl. Acad. Sci., USA, 87:9524-9528 (1990)). This single large polypeptide is co- or post-translationally processed to at least 9 polypeptides including two putative envelope proteins (E1 and E2/NS1) either by host signalase or viral proteases (see: Hijikata, M. et al., Proc. Natl. Acad. Sci., USA, 88:5547-5551 (1991); Hijikata, M. et al., Proc. Natl. Acad. Sci., USA, 90:10773-10777 (1993); Grakoui, A. et al., J. Virol., 67 (3): 1385-1395 (1993)). Hydropathy analysis of the deduced amino acid sequences of the HCV poly-protein showed the relatedness of HCV with pestiviruses and flaviviruses (see: Miller, R. H. and Purcell, R. H., Proc. Natl. Acad. Sci., USA, 87:2057-2061 (1990)).
In this regard, there have been several reports indicating that antibodies for the envelope glycoproteins of pestiviruses and flaviviruses (E1 and E2, E1 and NS1, respectively) protect their host against infection (see: Rumenapf, T. et al., J. Virol., 65 (2):589-597 (1991); Falgout, B. et al., J. Virol., 64 (9):4356-4363 (1990); Weiland, E. et al., J. Virol., 64 (8):3563-3569 (1990); Schlesinger, J. J. et al., J. Virol., 60 (3):1153-1155 (1986); Hulst, M. M. et al., J. Virol., 67 (9):5435-5442 (1993); Despres, P. et al., J. Gen. Virol., 72:2811-2816 (1991)). In the case of flavivirus, effective attenuated vaccines have been developed and more recently, recombinant based vaccines directed against flaviviruses and pestiviruses are under investigation (see: Rumenapf, T. et al., J. Virol., 65:589-597 (1991)). Further, two putative envelope glycoproteins (E1 and E2/NS1) of HCV, identified by in vitro processing analyses and N-terminal amino acid sequence analysis, have been expected to be the most probable components of preventive subunit vaccine considering that these may play a major role in the course of viral infection and host immune responses.
Accordingly, there is a need in the art for the development of a practical process for mass production of HCV E1 and E2/NS1 envelope glycoproteins for a diagnostic reagent and a potential preventive HCV vaccine.